a) Field of the Invention
The invention is directed to an optical arrangement with a light source for emitting a light bundle and with optical elements for transforming this light bundle into the shape of a light sheet, particularly suitable for illuminating individual planes of a three-dimensional specimen in selective plane illumination microscopy (SPIM).
b) Description of the Related Art
In contrast to confocal laser scanning microscopy (LSM), in which a three-dimensional specimen is scanned point by point in individual planes of different depth and the image information thus acquired is then combined to form a three-dimensional image of the specimen, the SPIM technique relies on widefield microscopy and makes it possible to display an image of the specimen based on optical sections through individual planes of the specimen.
The advantages of the SPIM technique include the greater speed at which image information is acquired, the reduced risk of bleaching biological specimens, and an expanded penetration depth of the focus in the specimen.
In principle, in the SPIM technique fluorophores which are by themselves contained in the specimen or have been introduced into the specimen for contrasting are excited by laser light, and the laser radiation is formed into a light sheet, as it is called. A selected plane in the depth of the specimen is illuminated by the light sheet and an image of this specimen plane is acquired in the form of an optical section by imaging optics.
To illustrate the geometry of the light sheet more clearly, it will be assumed in the context of the present invention that the light sheet has a cross section which extends in the X and Y coordinate directions perpendicular to the beam direction of the laser light and a length which extends in the Z coordinate direction along the beam direction.
The optical axis of the objective by which the illuminated specimen plane is to be imaged or observed is oriented perpendicular to the Z coordinate direction.
Optical arrangements for generating a light sheet in connection with the SPIM technique are described in DE 102 57 423 A1 and DE 10 2005 027077 A1.
These arrangements produce only a rigid light sheet which is not variable with respect to its thickness, which should correspond to the extension in the X coordinate. This circumstance is particularly disadvantageous when one and the same plane of a specimen is to be imaged successively in time with different objectives whose optical imaging characteristics differ from one another.
In such cases, it is desirable to have the possibility of adapting the geometry of the light sheet, above all, its thickness, to the respective objective so that only the plane of interest is actually illuminated in the specimen and, accordingly, an unwanted bleaching of the specimen substance outside this plane is prevented. Also, the depth of focus of the light sheet can be adapted in this way to the respective object field being observed.
Another disadvantage in the arrangements mentioned above is that the light sheet that is generated with them has a Gaussian intensity profile in cross section so that a uniform illumination of the specimen plane being examined is impossible.
WO 2004/0530558 A1 describes a method in which a light-sheet type illumination is generated based on a relative movement between a line-shaped light field and the specimen to be observed. The light-sheet type illumination is carried out in that the light field is lined up multiple times successively in time due to the relative movement. This has the disadvantage that shadows result within the plane of the specimen being examined because of portions of the specimen substance which are not transparent for the illumination light and which lie in the illumination direction.
EP 0 248 204 B1 describes the generation of a line-shaped illumination with a linear fiber array and cylindrical lenses arranged downstream. However, again, the geometry of the light sheet cannot be varied.
The publication U.S. Pat. No. 4,826,299 describes the shaping of a light sheet with a Powell lens. The Powell lens has an aspherical profile in one coordinate direction and is flat in the coordinate orthogonal to it so that a virtually homogenized line-shaped light field is formed from a light bundle and can be used as a light sheet. However, again the geometry of this light sheet can not be varied, specifically with respect to its thickness and length, so that an optimal illumination through the specimen plane to be examined is also impossible in this case when using different objectives whose optical characteristics differ from one another.
All of the arrangements mentioned above have the disadvantage of unwanted shadows within the plane of the specimen being examined which are caused by portions of the specimen substance in the illumination direction which are not transparent for the illumination light.